The goal of this project is to understand the anatomic and physiologic features of interneurons of the mammalian middle ear muscle (MEM) reflex pathway. The neurons of this reflex coordinate the activity of the MEMs to protect the inner ear from intense acoustic stimuli as well as reduce masking. This reflex arc is composed of primary auditory afferents originating in the cochlea, a single or series of interneurons originating in the cochlear nucleus and ultimately synapsing on MEM motoneurons, and efferent fibers of the facial and trigeminal nerves that terminate on the stapedius and tensor tympani muscles, respectively. Although features of primary auditory afferents and the motoneuron efferents have been well characterized, little is known about the reflex interneurons. Which subdivision of the cochlear nucleus contains the reflex interneurons? Is there a single or a series of interneurons from the cochlear nucleus to the facial and trigeminal nuclei? For Aim 1, we will perform focal lesioning studies of the cochlear nucleus using kainic acid, an excitatory neurotoxin. We will correlate focal lesioning of the cochlear nucleus with loss of the MEM electromyography (EMG) response, to determine which division of the cochlear nucleus is involved in the MEM reflex pathway. Since the anatomical cell types of the cochlear nucleus subdivisions are well known, these studies will narrow down the identity of the cochlear nucleus interneurons. For Aim 2, we will examine the cochlear nucleus interneurons by double-injection experiments. We will inject retrograde tracer into either the stapedius or tensor tympani muscles to label their respective motoneurons, and, at the same time, inject an anterograde tracer into the cochlear nucleus to label the interneurons of the MEM reflex. Injections of the cochlear nucleus will be guided by our lesion studies described in Aim 1. Labeled projections from the cochlear nucleus will be identified as interneurons of the MEM reflex if they terminate on labeled MEM motoneurons. Such terminations would reveal a direct connection between the cochlear nucleus and the MEM motoneurons. Overall, the proposed project will improve our understanding of the brainstem connections that comprise the MEM reflex pathway. These findings may provide a basis for refining and extending our interpretation of clinical tests of MEM reflex integrity and brainstem auditory processing in humans.